UNIVERSITY    OF    CALIFORNIA    PUBLICATIONS 

IN 

AGRICULTURAL    SCIENCES 

Vol.  1,  No.  1,  pp.    1-20  Issued  October  15,  1912 


THE  DISTRIBUTION  AND  ACTIVITIES  OP 

BACTERIA  IN  SOILS  OF  THE 

ARID  REGIONS^ 


BY 

CHARLES  B.  LIPMAN 


INTRODUCTION 

The  student  of  soils  in  the  humid  region,  when  for  the  first 
time  exploring  soils  in  the  arid  region,  is  invariably  struck  with 
the  extraordinary  depth  of  the  latter  as  against  the  very  shallow 
nature  of  the  former.  Taken  by  and  large,  and  excepting  the 
faulty  soils,  including  those  underlaid  at  no  great  depth  by  stiff 
clay,  coarse  gravel,  hardpan,  or  original  rock,  respective^,  the 
soils  of  the  arid  region  very  commonly  show  a  depth  of  at  least 
eight  to  ten  feet,  and,  when  viewed  in  section,  exhibit  such  a 
striking  uniform it.y  in  texture  and  color  as  to  attach  to  this 
unusual  condition,  in  the  mind  of  the  observer,  a  certain  marked 
practical  and  scientific  interest.  The  full  significance  to  crops  of 
the  arid  region  of  this  extraordinary  condition  in  our  soils  was 
first  realized  and  pointed  out  by  Hilgard  and  was  made  the 
subject,  by  him  and  Loughridge,  of  a  comprehensive  investiga- 
tion on  the  ''soil-columns"  of  California,  a  large  part  of  which 
is  completed,  but  some  of  which  is  still  in  progress.  The  study 
of  the  soil-columns  of  California  comprised  what  might  be 
looked  upon  as  a  very  thorough  partial  soil  survey  of  Cali- 
fornia. It  was  the  intention  of  the  investigators  above  named, 
at  the  inception  of  the  work,  to  obtain  columns  of  soil  repre- 
senting depths  of  twelve  feet,  including  a  sample  for  every  foot 


*  Eead    before    the    Society    of    American    Bacteriologists,    Washington, 
D.  C,  December  27,  1911. 


2  University  of  California  Publications  in  Agrindtural  Sciences    \  Vol.  1 

ill  depth,  and  to  obtain  a  kno\vled<iT  of  the  chemical  constitu- 
tion and  the  texture  of  the  soils  by  makino^  systematic  chemical 
and  mechanical  analyses  of  all  the  samples  thus  collected.  The 
information  thus  obtained  in  the  several  years  in  which  the  soil- 
columns  Avere  studied  by  Ililj^ard  and  Louo'hrid^e  and  the  large 
number  of  types  of  soils  considered,  alons:  Avith  the  most  striking' 
circumstance  of  the  depths  to  which  plant  root-systems  of  the 
arid  regions  penetrated,  led  Hilgard  to  believe  that  the  striking- 
chemical  and  mechanical  differences  between  the  soils  of  the  arid 
and  humid  regions,  as  well  as  the  differences  in  the  development 
of  the  root-systems  in  these  regions,  respectively,  might  find 
a  parallel  also  in  a  difference  between  the  bacterial  flora  at 
various  depths  in  the  soil.  It  was  this  belief  on  Hilgard's  part 
and  his  valuation  thereof  as  being  of  exceeding  scientific  interest 
as  well  as  practical  value,  that  led  to  the  association  with  him 
something  over  three  years  ago  of  the  writer,  and  it  was  then 
that  I  undertook,  among  other  biological  problems  in  soils,  a 
study  of  the  nitrogen-transforming  and  nitrogen-fixing  bacteria 
in  the  different  layers  of  soils  in  the  arid  region.  This  study. 
while  it  has  progressed  considerably,  is  still  in  the  first  stage 
of  its  development  and  the  complete  results  thereof  are  intended 
ultimately  to  be  combined  with  the  mechanical  and  chemical 
analyses  of  these  soils  in  a  comprehensive  report  on  the  whole 
work.  For  the  purposes  of  this  paper,  it  is  sufficient  to  give  a 
resume  of  some  of  the  important  results  obtained  in  these  inves- 
tigations with  an  account  of  the  methods  employed  in  the  work, 
so  that  it  may  serve  as  a  preliminary  communication  on  the  sub- 
ject and  bring  out  certain  striking  facts  with  reference  to  the 
distribution  of  bacteria  in  California  soils. 

:\rp7rTT0T)s  employp]d  in  these  investigations 

One  of  the  most  difficult  problems  in  connection  with  these 
investigations  was  to  find  a  method  for  the  collection  of  soil 
sam[)les  at  the  several  depths  which  would  fairly  represent  the 
actual  conditions  which  obtain  there,  so  far  as  the  bacterial 
Hora  are  concerned.  Our  first  attempts  in  this  direction  were 
made  with  an  auger  of  the  typc^  manufactured  by  Iwan  Brothers 
at  South   Bend,  Indiana,  by  means  of  which  we  tried,  through 


1912]  Lipman:  Bacteria  in  Soils  of  Arid  Eegions  3 

successive  sterilization  of  the  auger  (before  taking  each  sample), 
to  obtain  a  sample  which  represented,  uncontaminated,  each  of 
the  soils  as  they  are  found  in  their  natural  state  in  the  field. 
With  a  sterile  spatula  there  were  taken  from  the  samples  thus 
obtained  with  the  auger  representative  samples  which  were  im- 
mediately placed  in  sterile  cotton-stoppered  bottles.  It  was 
soon  found,  however,  that  this  method  could  not  be  relied  upon 
for  accurate  results,  since  no  matter  how  carefully  the  samples 
were  thus  taken,  there  were  many  chances  for  contaminating 
samples  from  the  lower  layers  with  soil  from  the  upper  layers 
and  thus  obtaining  results  which  were  erroneous.  After  much 
experimenting  we  finally  decided  on  the  following  plan  for 
taking  the  soil  samples,  which,  so  far  as  I  know,  is  as  free  from 
chances  of  error  as  any  method  that  can  be  adopted  in  a  series 
of  investigations  which  must  of  necessity  be  so  extensive.  In- 
deed, I  believe  the  chances  for  error  here  are  so  small  that  they 
cannot  affect  the  validity,  to  any  appreciable  extent,  of  the 
results  obtained.  Our  method  consists  in  having  dug,  a  day 
or  two  prior  to  sampling,  a  hole  twelve  feet  in  depth  with  at  least 
one  vertical  wall  and  large  enough  for  a  man  to  stand  in.  The 
samples  are  taken  as  follows:  With  a  sterilized  spade,  a  layer 
of  soil  of  about  five  or  six  inches  in  depth  is  sliced  down  along 
the  whole  length  of  the  wall  which  is  to  be  sampled.  After  this 
is  done,  to  remove  the  soil  that  in  the  one  or  two  days'  exposure 
may  have  become  contaminated,  the  fresh  surface  thus  obtained 
on  the  vertical  wall  is  sterilized  by  means  of  a  plumber's  torch 
on  the  surface  surrounding  every  spot,  previously  marked  off, 
at  which  a  sample  from  each  foot  in  depth  is  to  be  taken.  When 
this  is  done  a  sterile  cylindrical  tin  tube,  a  little  over  one  inch 
in  diameter  and  about  ten  inches  long,  is  driven  at  right  angles 
to  the  wall  into  the  spot  selected  for  sampling,  immediately 
drawn  out  when  sufficient  soil  has  thus  been  obtained,  and  the 
cotton  plug  replaced.  In  our  first  experiments,  glass  tubes  of 
the  size  described  were  employed,  with  paraffined  corks  at  one 
end  and  cotton  stoppers  at  the  other.  We  found  this  to  be  a 
poor  method,  however,  and  have  replaced  the  glass  by  tin  tubes, 
closed  at  one  end  and  plugged  with  cotton  at  the  other.  These 
are  sterilized  at  150  deg-rees  centierrade  for  one  hour  and  a  half 


4  University  of  California  Publications  in  Agricultural  Sciences    [Vol.  1 

before  using.  In  this  way  by  the  use  of  a  plumber's  torch  at 
every  depth  as  we  descend  from  the  surface  of  the  soil  down 
to  the  twelve-foot  depth,  we  obtain,  by  starting  at  sterile  sur- 
faces, a  sample  of  soil  representing  as  nearly  as  possible  the  true 
condition  which  obtains  at  very  depth.  The  samples  are  marked 
properly,  taken  to  the  laboratory,  and  examined  for  their  am- 
monifying, nitrifying  and  nitrogen-fixing  powers  by  means  of 
a  modified  Remy  method,  the  solutions  employed  for  the  work 
being  prepared  in  accordance  with  the  formulae  used  by  J.  G. 
Lipman.^  Every  50  c.c.  portion  of  the  medium  in  a  250  c.c. 
Erlenmeyer  flask  is  inoculated  with  5  grams  of  soil. 

DESCRIPTION    OF    SOILS    EMPLOYED    IN    THESE 
EXPERIMENTS 

The  descriptions  given  below  represent  the  soils  which  were 
employed  for  bacteriological  examinations  and  sampled  for  the 
purpose  as  above  described.  The  numbers  employed  below  are 
used  throughout  all  the  following  tables  so  as  to  make  unneces- 
sary any  further  descriptions. 

Soil  No.  1.  Eed  clay  loam  mesa  soil,  from  Riverside,  California,  on 
which  good  orange  trees  were  growing  at  time  of  sampling.  The  soil  is 
well  supplied  with  potash,  but  rather  poor  in  phosphoric  acid  and  very 
poor  in  humus  and  nitrogen.  It  is  underlaid  by  hardpan  at  six  feet  from 
the  surface,  which  continues  on  down  to  the  twelve-foot  depth,  "With  the 
careful  cultivation  which  is  given  it,  along  with  proper  fertilization  and 
tillage,  the  soil  produces  profitable  crops  of  oranges  and  lemons. 

Soil  No.  2.  Silty  alluvial  loam,  from  Davis,  California.  The  samples 
used  were  obtained  from  between  some  fig  trees  at  the  University  Farm. 
This  soil  is  practically  uniform  in  color  from  the  first  foot  to  the  twelfth 
and  only  becomes  slightly  different  in  texture  below  the  fifth  foot,  becoming 
gradually  coarser  and  sandier  as  we  descend  to  the  lower  layers.  It  is 
well  supplied  with  potash,  phosphoric  acid,  and  lime  and  has,  for  a  soil  of 
the  arid  region,  a  normal  content  of  humus. 

Soil  No.  3.  Sandy  alluvial  loam,  from  Davis,  California.  Samples  were 
taken  from  a  wheat  field  at  the  University  Farm,  only  to  a  depth  of  ten 
feet.  This  soil  is  well  supplied  with  j)hosphoric  acid,  potash  and  lime, 
but  rather  poor  in  humus  and  nitrogen.  The  sand  is  of  a  coarse  nature 
and  becomes  rapidly  coarser,  descending  from  the  first  foot  down  to  the 
twelfth,  where  it  is  found  as  very  coarse  sand. 


1  Bulletin  180.     N.  J.  Agr.  Expt.  Station. 


1912]  Lipmau:  Bacteria  in  Soils  of  Arid  Eegions  5 

Soil  No.  4.  Sandy  alluvial  loam,  from  Davis,  California.  Samples  ob- 
tained in  almond  orchard  at  the  University  Farm.  This  soil  is  not  nearly 
so  coarse  as  soil  No.  3  and  shows  a  more  uniform  texture  throughout  a 
seven-foot  depth,  but  after  that  becomes  coarser  in  texture.  It  is  better 
supplied  with  humus  and  nitrogen  than  soil  No.  3  and  is  well  supplied 
with  potash,  phosphoric  acid,  and  lime. 

Soil  No.  5.  Alluvial  loam,  from  Davis,  California.  Samples  obtained 
in  a  pear  orchard  at  the  University  Farm.  The  soil  is  uniformly  of  a 
fine  sandy  loam  texture  for  a  depth  of  nearly  five  feet  and  then  rapidly 
becomes  much  coarser  than  the  soil  at  similar  depths  in  No.  3.  The  upper 
soil  is  well  supplied  with  potash,  phosphoric  acid,  and  lime,  and  fairly  well 
supplied  with  humus  and  nitrogen.  The  lower  layers  are  rather  poor  in 
phosphoric  acid,  humus  and  nitrogen. 

Soil  No.  6.  Fine  silty  soil,  from  Hanford,  California.  Samples  taken 
from  a  vineyard  at  Hanford,  from  the  first  to  the  ninth  foot  only.  No 
sampling  was  done  below  the  ninth  foot  because  of  the  fact  that  the  water- 
table  was  reached  at  about  that  point  and  it  was  almost  impossible  to  get 
samples  uneontaminated.  This  soil  is  almost  devoid  of  humus  and  contains 
but  little  nitrogen,  but  is  fairly  well  supplied  with  phosphoric  acid,  potash, 
and  lime.     No  alkali  is  present  in  the  soil. 

Soil  No,  7.  Silty  alluvial  loam,  from  Davis,  California.  Samples  taken 
at  the  University  Farm,  close  to  a  young  eucalyptus  tree,  about  twenty 
months  old.  The  soil  is  fairly  uniform  in  texture  throughout  the  entire 
depth  studied  and  is  fairly  well  supplied  Avith  humus  and  nitrogen,  and 
well  supplied  with  phosphoric  acid,  potash,  and  lime.     No  alkali  is  present. 

Soil  No.  8.  Alkali  soil  from  Tulare,  California.  Taken  only  to  a  depth 
of  ten  feet,  owing  to  water  conditions  such  as  those  described  in  soil  No.  6. 
This  soil  contains  very  little  humus  and  is  strongly  impregnated  with  salts, 
especially  ' '  black  alkali. "  It  is  otherwise  well  supplied  with  phosphoric 
acid,  potash,  lime,  and  the  other  minerals.  Hardly  any  vegetation  can  exist 
on  this  soil  after  the  salts  have  risen  to  the  upper  layers. 

Soil  No.  9.  A  very  stiff  and  tenacious  silty  clay  adobe,  from  Imperial, 
California.  Uniform  in  texture  from  the  surface  down  to  the  eighth  foot, 
at  which  there  is  found  a  layer  of  fine  sand  for  a  foot  and  a  half  in 
depth  and  then  a  silty  sand  below  to  the  twelve-foot  depth.  The  soil 
throughout  is  almost  devoid  of  humus  and  contains  but  very  little  nitrogen; 
It  is  very  rich,  however,  in  phosphoric  acid,  potash,  and  lime.  The  upper 
layers  of  the  soil  consist  of  particles  of  silt  and  clay  which  are  so  fine 
as  to  become  cemented  together  into  an  extremely  hard,  refractory  material, 
which  is  almost  of  the  consistency  of  a  dry,  but  not  heated,  brick.  A 
considerable  quantity  of  common  salt  is  present  in  this  soil.  This  soil  has 
never  been  cultivated  or  cropped. 

Soil  No.  10.  Fine,  sandy  soil  from  the  desert  of  Coachella  Valley.  In 
this,  as  in  the  Imperial  Valley  soils,  there  are  to  be  found  narrow  layers 
of  an  inch  or  two,  and  sometimes  more,  of  very  fine  shells  of  former  life 
which  existed  in  the  water  at  one  time  covering  this  land.     The  soil  has  very 


6  University  of  California  Publications  in  Agricultural  Sciences    [Vol.  1 

little  or  no  humus  and  nitrogen.  It  is,  however,  rich  in  phosphoric  acid  and 
lime  and  -well  supplied  with  potash.  The  soil  is  uniform  in  texture  through- 
out the  twelve-foot  depth  and  becomes  only  a  little  coarser  at  twelve  feet. 
The  only  changes  visible  in  color  and  texture  in  the  vertical  wall  are 
merely  those  of  the  shell  layers  above  noted.  The  soil  from  which  these 
samples  w^ere  taken  has  never  been  cropped,  but  similar  soil,  with  a  good 
water  supply,  produces  very  fine  alfalfa.  Very  little  alkali  is  present  in 
this  soil. 

Soil  No.  11.  Fertile,  alluvial  loam  from  Hayw^ard,  California.  Uniform 
in  texture  for  seven  feet  and  then  rapidly  becoming  quite  coarse  and  re- 
maining so  down  to  a  depth  of  twelve  feet.  This  soil  is  very  fertile, 
producing  good  crops  of  cherries,  walnuts,  potatoes,  and  other  agricultural 
plants.  It  is  well  supplied  with  humus  and  nitrogen,  judged  by  the 
standard  for  soils  of  the  arid  region,  throughout  the  twelve-foot  depth. 
The  phosphoric  acid,  potash,  and  lime  are  also  plentiful  in  all  the  soil  layers. 
The  samples  used  in  these  investigations  were  obtained  in  a  cherry  orchard. 

AMMONIFICATION  IN  SOIL  COLUMNS 

Second  only  to  the  importance  of  soil  bacteria  in  maintaining 
the  total  nitrogen  supply  in  soils  is  their  power  to  supply  con- 
stantly available  nitrogen  to  plants.  The  essential  nature  of 
this  important  phase  of  the  activities  of  soil  organisms  is  in  no 
wise  detracted  from  by  the  recent  research  which  has  made  it 
clear  that  some  plants  at  least  can  take  their  nitrogen  from  the 
soil  in  forms  other  than  the  nitrate.  While  many  of  them  may 
not  absorb  their  nitrogen  in  the  form  of  nitrates,  it  seems  quite 
certain  that  practically  all  of  them  must  take  their  nitrogen  in 
forms  much  simpler  than  the  proteid.  This  being  undeniably 
the  case,  some  agency  in  the  soil  is  necessary  to  accomplish  the 
transformation  of  the  organic  nitrogen  (no  matter  what  the 
source  of  the  latter  to  the  soil  may  be)  into  a  simpler,  more 
available,  or  more  assimilable  form.  These  agencies  we  have 
found  to  be  the  various  types  of  soil  organisms  which  constitute 
what  we  now  designate  by  the  term  "ammonifying  flora"  of  the 
soil. 

With  these  statements  admitted,  it  seems  reasonable  to  sup- 
pose that  any  increase  in  the  activities  of  the  organisms,  included 
under  this  head,  is  a  distinct  advantag^e  to  the  plant.  Under  our 
climatic  conditions,  where,  as  above  stated,  the  plant  roots  very 
deeply,  besides  making  a  large  lateral  root-development,  it  is 
necessary  to  have  the  activities  of  the  ammonifying  organisms 


1912]  Lipman:  Bacteria  in  Soils  of  Arid  Regions  7 

not  only  in  the  upper  layers  of  the  soil,  but  in  the  lower  layers 
where  an  actual  examination  of  the  root-systems  of  plants  shows 
a  large  development  of  fibrous  or  feeding  roots.  A  study,  there- 
fore, of  the  ammonifying  powers  of  the  different  layers  of  soil, 
or,  rather,  of  the  microorganic  flora  which  they  contain,  is  of 
practical  moment,  since  it  is  bound  to  throw  light  on  the  soluble 
nitrogen  supply  for  roots  in  the  greater  depths  of  soil  and 
indicate  what  practical  measures  may  be  taken  toward  sustain- 
ing and  encouraging  the  growth  and  activities  of  the  organisms 
responsible  for  that  soluble  nitrogen  supply.  Since,  therefore, 
we  assume  ammonia  production  to  be  the  first  great  step  recog- 
nized by  our  analytical  methods  in  the  transformation  of  soil 
nitrogen,  I  have  first  determined  the  ammonifying  powers  at 
various  depths  of  soils,  which  may  be  considered  typical  of  well- 
defined  areas  and  conditions  in  the  arid  region. 

For  this  purpose  there  were  inoculated  into  sterile  50  c.c.  por- 
tions of  1  per  cent  peptone  solution,  5  grams  of  soil  from  every 
foot  from  the  surface  down  to  the  last  depth  taken,  as  above 
described.  After  four  days  incubation  at  about  28  degrees 
centigrade,  the  cultures  were  washed  into  copper  distilling  flasks, 
sufficient  distilled  water  added,  as  well  as  a  slight  excess  of 
magnesia,  and  distilled.  The  distillate  was  caught  in  standard 
tenth  normal  hydrochloric  acid,  the  excess  of  which  was  titrated 
with  standard  tenth  normal  ammonia.  Table  I  gives  the  results 
of  determinations  of  the  ammonifying  power  of  the  soils  chosen, 
as  above  described.  The  ammonifying  power  of  only  one  soil, 
namely  No.  6,  is  not  given,  for  the  reason  that  the  soil  column 
had  inadvertently  become  contaminated  before  we  were  ready 
to  use  it. 

The  numbers  of  the  soils  refer  to  corresponding  numbers 
under  the  descriptions  given  above,  and  the  amounts  of  ammonia 
produced,  as  given  in  the  table,  represent  milligrams  of  nitrogen 
as  ammonia. 

The  data  given  in  table  I  prove  very  clearly  two  facts.  First, 
that  in  the  typical  deep  and  normal  soils  of  the  arid  region,  the 
activities  and  the  distribution  of  the  ammonifying  flora  seem  to 
run  parallel  with  the  texture,  the  chemical  composition,  and  the 
root-development  in  these  soils.     Second,  that  in  the  absence  of 


University  of  California  Publications  in  Agricultural  Sciences    [Vol.  1 


TABLE 

I 

Ammonification  in 

Soil  Columns 

Soil  No,  1 

2 

3 

4 

5 

6          7 

8 

9 

10 

11 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

1st  ft. 

59.80 

67.76 

68.25 

61.60 

54.67 

72.05 

7.28 

25.46 

12.15 

42.84 

2nd  ft. 

55.46 

77.00 

72.66 

54.46 

49.14 

68.90 

4.90 

8.27 

6.46 

36.84 

3rd  ft. 

52.30 

69.02 

68.40 

38.50 

32.76 

70.43 

5.46 

8.20 

3.21 

14.14 

4th   ft. 

55.83 

70.63 

43.05 

44.80 

lost 

65.72 

2.80 

8.69 

1,99 

7.28 

5th   ft. 

49.72 

67.48 

34.23 

39.90 

8.40 

63.69 

4.76 

4.87 

1,25 

7.84 

6th  ft. 

49.00 

84.63 

31.64 

47.60 

15.68 

60.50 

4.41 

8.27 

1,61 

21.19 

7th  ft. 

31.70 

72.17 

42.00 

44.80 

17.85 

56.90 

2.45 

6.03 

1,57 

7.14 

8th  ft. 

30.54 

52.57 

35.00 

22.54 

9.80 

50.43 

10.22 

5.40 

1.34 

7.48 

9th  ft. 

28.65 

52.43 

35.70 

32.90 

15.40 

45.69 

10.64 

2.18 

1,24 

5,60 

10th  ft. 

25.40 

36.89 

29.40 

10.22 

43.25 

2.23 

2,11 

16,38 

11th  ft. 

15.65 

21.56 

25.48 

11.06 

40.76 

1.34 

4.87 

16,64 

12th  ft. 

14.30 

35.84 

38.22 

10.50 

38.45 

2.62 

1.44 

10,85 

humus  and  moisture,  or  in  the  presence  of  alkali  salts,  the  activi- 
ties of  ammonifying  organisms  are  seriously  handicapped. 

To  discuss  these  more  in  detail  we  find,  for  example,  in  soil 
No,  1,  derived  from  the  mesa  soil  at  Arlington  Heights,  River- 
side, a  strong  ammonification,  varying  but  little  from  the  first 
foot  down  to  the  seventh,  below  which  depth  we  find  a  sudden 
marked  decrease  in  ammonia  production,  for  the  reason,  doubt- 
less, that  from  the  sixth  foot  down  to  the  twelfth  we  find  a 
layer  of  hardpan  which,  owing  to  its  poor  aeration  and  poor 
water  conditions,  is  unfit  for  the  development  of  a  vigorous  bac- 
terial flora.  In  other  words,  we  find  in  this  soil-column,  through 
the  ammonifying  power  of  the  various  depths  of  soil,  an  expres- 
sion of  the  vigor  and  numbers  of  bacteria  present  in  these  soil 
layers  and  also  of  the  amounts  of  soluble  nitrogen  which  can 
there  be  expected  to  be  made  available  through  the  agency  of 
soil  organisms. 

In  soil  No,  2,  however,  which  represents  a  good,  deep  alluvial 
soil,  we  find  a  very  vigorous  ammonification  from  the  first  seven 
feet,  and  only  slightly  reduced  ammonia  production  in  the  eighth 
and  ninth  feet,  after  which  we  find  a  large  reduction  of  about 
50  per  cent  in  ammonia  production  for  the  other  three  feet. 
We  have  here,  therefore,  good  vigorous  ammonia  production  down 


1912]  Lipman:  Bacteria  in  Soils  of  Arid  Regions  9 

to  the  tenth  foot  and  therefore  an  indication  that  in  these  soils 
there  is  constantly  being  made  available  nitrogen,  if  organic 
nitrogen  be  present  from  humus  and  other  sources,  for  the  needs 
of  plants  with  deep  root  systems. 

In  soil  No.  3,  which  is  more  sandy  than  the  other  alluvial 
soil  described  and  which  rapidly  becomes  coarser  in  texture  as 
we  descend  into  the  lower  layers,  we  find  vigorous  ammonifica- 
tion  to  obtain  down  to  the  fourth  foot,  below  which  we  find  a 
considerable  decrease  in  ammonifying  power,  owing  to  the  fact 
that  in  that  coarse  soil  neither  water  nor  humus,  nor  soluble 
minerals,  are  present  in  sufficient  quantity  to  encourage  bacterial 
development.  Here,  however,  we  find  the  general  tendency  for 
ammonifying  organisms  to  penetrate  to  the  greater  depth  quite 
plainly  visible.  The  remarks  made  for  soils  2  and  3  are  just 
as  truly  applicable  to  the  other  alluvial  soils  from  the  same 
district  represented  by  Nos.  4,  5,  and  7.  The  marked  produc- 
tion of  ammonia,  even  in  the  twelfth  foot  of  No.  7,  is  in  accord 
with  the  fine  physical  and  chemical  condition  of  that  soil  to 
that  depth  and  therefore  deserves  additional  mention  here. 

As  to  other  types  of  soils,  the  data  in  the  table  show  plainly 
enough  what  a  profound  effect  strong  alkali  salts  (both  black 
and  white  alkali  salts  among  them)  may  exert  on  the  ammoni- 
fying flora  and  their  vigor.  Here  ammonification  is  indeed  very 
feeble  in  the  surface  soil,  becoming  feebler  as  we  go  down  until 
the  eighth  foot  is  reached,  at  which  depth,  as  well  as  in  the  ninth 
foot,  we  find  quite  a  marked  increase  in  ammonia  production. 
This  is  doubtless  due  to  the  fact  that  the  total  salt-content  is 
at  that  depth  much  lower  and  therefore  not  so  seriously  affecting 
the  activities  of  the  organisms  there  contained.  As  for  the 
desert  soils,  which  never  have  contained  much  humus  and  very 
frequently  contain  too  much  alkali,  it  is  natural  to  expect  a 
rather  feeble  ammonifying  power  on  the  part  of  the  soils.  Table 
I  shows  that  in  this  case  the  expected  happens.  In  soil  No.  9. 
for  example,  not  only  the  lack  of  humus  and  moisture,  but  the 
very  unfavorable  physical  condition,  above  referred  to  under 
the  description  of  that  soil,  along  with  its  salt-content,  have  so 
far  affected  the  ammonifying  power  of  that  soil  as  to  reduce  it 
to  a  little  over  one-third  of  what  the  normal  vallev  soils  de- 


10  University  of  California  Publications  in  Agricultural  Sciences    [Vol.  1 

scribed  have  exhibited.  Moreover,  it  would  seem  that  the  salt- 
content  in  the  lower  layers  of  this  soil,  which  increases  as  we 
go  down,  has  very  seriously  checked  the  development  of  these 
organisms  there  and  was  probably  assisted  by  the  unfavorable 
physical  condition  mentioned.  In  soil  No.  10,  while  the  salt- 
content  is  only  meager,  we  have  a  rather  coarse,  sandy  soil  with 
hardly  any  humus,  which  is  therefore  for  that  reason  an  unfavor- 
able medium  for  the  development  of  bacteria,  to  say  nothing  of 
the  lack  of  moisture  there  and  the  great  heat  which  these  desert 
soils  must  absorb  from  the  sun.  We  therefore  have  a  very  much 
smaller  ammonifying  power  in  the  upper  layers  of  the  soil  than 
exists  even  in  soil  No.  9,  from  Imperial,  and  then  a  very  rapid 
decrease  to  almost  no  ammonifying  power  in  the  lower  layers. 

By  a  general  survey  of  all  of  these  data,  it  would  certainly 
seem  that  we  are  justified  in  drawing  the  conclusion  that  am- 
monification  and  the  ammonifying  flora  of  soils  are  vigorous  for 
several  feet  down  in  the  arid  region  and  are  limited  in  their 
activities  only  by  the  presence  of  large  amounts  of  salt  or  a 
lack  of  humus  and  moisture.  Since,  however,  California  soils, 
taken  by  and  large,  are  deep,  we  have  reason,  from  the  facts 
above  given,  to  suppose  that  the  ammonifying  power  in  most 
of  these  soils,  which  are  not  in  any  way  "abnormal,"  is  vigorous 
at  great  depths. 

NITRIFYING   POWERS   OF    SOIL   COLUMNS 

By  very  many  and  perhaps  by  most  plants,  nitrate  is  the 
form  of  nitrogen  taken  up.  It  is  therefore  of  importance  not 
only  to  study  ammonia  formation  in  soils,  but  nitrate  formation 
as  well.  In  these  investigations  we  have  studied  qualitatively 
and  quantitativel}^  the  production  of  nitrites  and  nitrates  in 
ammonium  sulfate  solution  by  soils  from  the  different  depths 
in  every  case.  Here  also,  as  in  the  ammonification  work,  5  grams 
«of  soil  were  used  to  inoculate  50  c.c.  of  culture  solution.  The 
results  obtained  in  this  work  are  set  forth  in  a  qualitative  man- 
ner, as  to  nitrate  formation  merely,  in  table  II,  since  it  is 
sufficient  for  the  purpose  of  this  preliminary  paper  to  know  to 
what  depths  in  the  soil  nitrates  are  produced.  Later  publica- 
tions, giving  the  more  complete  data  of  these  investigations,  will 


1912]  Lipman :  Bacteria  in  Soils  of  Arid  Begions  11 

give  the  quantitative  results  as  they  are  given  for  ammonification 
in  table  I.  The  plus  sign  represents  nitrate  formation  and  the 
minus  sign  the  absence  thereof.  The  numbers  of  the  soils  are 
referred  again  to  the  descriptions  above  given. 

.     TABLE  II 

Nitrification  in  Soil  Columns 

Soil  No.    1         2         3         4         5         6         7         8         9        10       11 

1st   ft.        +        +        +        +        +       +        +       ___       + 

2nd  ft.  +  +  +  +  +  —       +       —       —       —  + 

3rd  ft.  +  +  +  +  +  _       +       ___  + 

4th  ft.  +  +  +  +  +  _       +       ___  + 

5th  ft.  +  +  +  +  +  _____  + 

6th  ft.  trace  +  —  —  +  —       ___       —  + 

7th  ft.  —  —  —  —  —  —       —       —       —       —  J^ 

8th  ft.  _  —  _______       —  + 

10th  ft.  —  —  —  —  —  ___ 
11th  ft.  —  —  —  —  —  ___ 
12th  ft.   —   —       _—       _       ___ 

From  the  data  in  table  II  we  see  again  a  striking  resemblance 
between  nitrification  in  the  soil  depths  and  ammonification  in 
the  same.  All  the  alluvial  soils  in  particular  show  very  uniform 
nitrate  formation.  The  latter  seems  to  be  as  much  inhibited 
in  soil  No.  1  by  the  hardpan  layer  as  is  ammonification.  In 
soils  2,  3,  4,  and  5,  as  well  as  7,  we  find  a  general  tendency  for 
nitrates  to  be  formed  in  the  first  five  feet  and  then  an  enfeebled 
powTr  of  nitrate  formation,  in  some  cases  for  one  foot  and  in 
other  cases  a  total  loss  of  that  power.  In  nearly  all  cases  these 
run  parallel  with  a  similar  decrease  in  ammonia  formation,  but 
it  seems  that  nitrate  formation  is  more  seriously  hampered  by 
the  conditions  which  curtail  ammonia  formation  and  particularly, 
it  appears,  by  the  lack  of  oxygen  in  the  lower  layers  of  the  soil. 
This  is  an  account,  therefore,  of  the  first  case  which  has  come  to 
my  notice  of  nitrification  at  any  depths  below  two  or  three  feet 
in  the  soil,  and  shows  a  marked  difference  in  itself  between  soils 
formed  and  existing  under  humid  and  those  formed  and  exist- 
ino:  under  arid  conditions.     Nitrogen  therefore  is  available  in 


12  University  of  California  Publications  in  Agricultural  Sciences    [Vol.  1 

these  soils,  not  only  for  those  plants  which  are  able  to  absorb 
ammonia  nitrogen,  but  also  for  that  larger  class  of  normal 
plants  which  absorb  their  nitrogen  in  the  nitrate  form.  It  must 
be  said  here,  however,  that  nitrate  formation  proceeded  always 
more  rapidly  in  soils  from  the  first  foot  than  in  cultures  pre- 
pared from  the  other  depths.  This  may  indicate  a  smaller  num- 
ber of  nitrifying  organisms  in  the  lower  layers  of  the  soil  or 
perhaps  a  less  vigorous  flora,  but  their  activities  are  uniform 
from  the  second  foot  down  to  the  last  depth  in  which  they 
show  no  activity  as  indicated  in  table  II.  In  soil  No.  6,  which 
w^e  find  on  analysis  contained  merely  a  trace  of  humus,  that  cir- 
cumstance seems  to  have  made  the  soil  unfit  for  the  develop- 
ment of  the  vigorous  bacterial  flora  and  is  supported  by  the 
data  in  tables  II  and  III.  As  to  the  nitrate  formation  in 
culture  solutions  by  the  inoculation  with  this  Hanford  soil, 
nitrates  were  produced  only  after  a  month's  incubation  and  only 
in  small  quantities  in  the  culture  prepared  from  the  upper  foot 
of  soil,  whereas  all  other  surface  soils,  Avhen  inoculated  into  solu- 
tions with  the  exception  of  those  which  show  no  nitrification  at 
all,  showed  nitrate  formation  before  the  end  of  two  weeks.  In 
culture  solutions  from  soil  No.  6,  kept  about  three  months  and 
prepared  from  the  lower  layers  of  soil,  no  nitrates  were  ever  to 
be  found.  The  depressing  effect  of  alkali  on  the  bacterial  flora, 
as  well  as  the  inhibiting  effect  of  a  lack  of  humus,  moisture, 
and  the  proper  physical  condition,  are  again  exemplified  in 
table  II  in  soils  8,  9,  and  10,  as  they  w^ere  for  the  same  soils  in 
table  I  referring  to  ammonification.  Even  after  one  month's 
incubation,  not  one  of  these  soil-samples  showed  any  nitrate 
formation,  whether  the  culture  was  prepared  with  the  soil  from 
the  upper  layers  or  from  the  lower.  There  seems  to  be  a  total 
absence  of  nitrifying  bacteria  of  one  kind  or  another. 

The  best  example  of  the  penetration  of  nitrifying  bacteria 
to  great  depths  was  obtained  in  soil  No.  11,  a  fine  alluvial  loam 
from  Hayward,  where  nitrate  formation  was  obtained  down  to 
the  ninth  foot  in  the  soil.  In  this  case  also  there  was,  besides 
a  mere  formation  of  nitrates,  as  shown  by  a  qualitative  test,  an 
actually  vigorous  nitrate  formation  in  the  lower  layers  as  well 
as  in  the  upper  layers  of  the  soil.     It  would  seem  again  here 


1912]  Lipman :  Bacteria  in  Soils  of  Arid  Regions  13 

therefore,  in  general,  that  where  soils  in  the  arid  region  are 
supplied  with  a  moderate  amount  of  humus,  with  the  proper  tex- 
ture and  chemical  constitution,  as  well  as  freedom  from  alkali, 
all  of  which  is  true  of  the  large  majority  of  our  soils,  nitrification 
as  well  as  ammonification  is  found  to  obtain  vigorously  in  the 
lower  layers  of  the  soil  for  four  feet  at  least,  and  in  some  cases 
to  six  and  to  nine  foot  depths. 

NITROGEN  FIXATION  IN  SOIL  COLUMNS 

The  next  point  of  interest  to  determine  in  these  soil-column 
investigations  from  the  bacteriological  standpoint  was  to  show 
whether  or  not  the  supply  of  nitrogen,  at  the  disposal  of  the 
ammonia-forming  and  nitrate-forming  organisms,  which  we  have 
found  developed  to  such  great  depths,  and  enabling  roots  to 
have  a  soluble  nitrogen  supply  there,  was  provided  merely  by 
the  humus  content  of  the  soil  at  those  depths  and  produced  from 
decaying  roots,  or  carried  dow^n  from  the  upper  layers;  or,  was 
that  nitrogen  supply  in  part  a  new  one  obtained  directly  from 
the  atmosphere  by  nitrogen-fixing  bacteria.  If  such  were  the 
case,  we  should,  of  course,  have  enormous  quantities  of  nitrogen 
fixed  per  acre,  since  the  fixation  would  not  be  limited  to  the 
upper  foot  of  soil.  Accordingly,  experiments  were  inaugurated 
to  obtain  the  facts  which  exist  with  reference  to  this  matter. 

Here  the  necessary  mannite  solution  w^as  inoculated  with  five 
grams  of  soil  in  each  case,  and  a  culture  prepared  from  every 
foot  in  depth  in  the  case  of  every  soil.  Table  III  shows  in 
tabular  form  the  results  obtained,  which  are  set  forth  qualita- 
tively. The  numbers  at  the  heads  of  the  columns  refer  again 
to  the  numbers  used  in  the  description  of  soils,  and  one  plus 
sign  is  intended  to  show  the  presence  of  Azotobacter,  two  of 
a  fairly  vigorous  development  of  these  organisms,  and  three  of 
a  very  vigorous  development.  In  this  qualitative  way,  there- 
fore, nitrogen  fixation  has  been  judged  by  the  development  of 
Azotobacter  as  a  criterion.  It  may  justly  be  argued  against  this 
that  other  organisms  are  capable  of  fixing  nitrogen  and  that  the 
quantitative  figures  would  be  preferable  to  the  qualitative  one 
showing  merely  the  presence  of  Azotobacter.  While  this  argu- 
ment may  in  part  be  true,  it  appears  from  my  results,  which 


14  University  of  California  Fuhlications  in  Agricultural  Sciences    [Vol.  1 

show  quantitative  as  well  as  qualitative  figures  in  these  as  well 
as  other  experiments,  that  in  the  absence  of  Azotobacter  only 
very  slight  fixations  of  nitrogen  or  none  are  obtained. 

From  the  results  set  forth  in  table  III,  it  appears  that  only 
one  soil  of  the  eleven  tested  shows  the  presence  of  Azotobacter 
as  deep  down  as  the  fourth  foot  and  six  others  show  the  pres- 
ence of  these  organisms  in  the  third  foot.  Most  of  them,  how- 
ever, show  the  presence  of  Azotobacter  in  vigorous  form  only 
in  the  first  two  feet.    It  is  therefore  not  sufficient,  evidently,  for 

TABLE  III 

Nitrogen  Fixation  in  Soil  Columns 

Soil  No.     1  2             3  4  5  6  7  8  9  10  11 

1st  ft.  +H-+  +  +  +  +  +  +  +  +  +  +++  -  +  +  +  —  —  —  +  + 

2ndft.  ++  +  +  +  +  +  +  +  +  +  +  +  +  —  +  +  +  —  —  —  +  + 

3rd  ft.  +  —  ++  +  +  +  +  +  +  —  ++  —  —  —  +  + 

4th  ft.  —  —  —  —  +  +  +  —  —  —  —  —  + 

5th  ft.  —  —  —  —  —  —  —  _  _  _  _ 

6th  ft.  —  —  —  —  —  —  —  -.  _  _  _ 

7th  ft.  —  —  —  —  —  —  —  _  _  _  _ 

8th  ft.  —  —  —  —  —  —  —  _  _  _  _ 

9th  ft.  —  —  —  —  —  —  —  _  _  _  _ 

10th  ft.  —  —  —  —  —  —  —  —  —  —  — 

11th  ft.  —  —  —  —  —  —  —  —  —  —  — 

12th  ft.  —  —  —  —  —  —  —  ____ 

soils  to  be  chemically  and  physically  as  favorably  constituted  as 
these  soils  are  for  ammonification  and  nitrification  to  encourage 
the  deeper  penetration  of  Azotobacter.  As  is  well  known,  these 
organisms  are  extremely  sensitive  to  a  lack  of  oxygen  and  it 
would  appear  that  this  circumstance  regulates  and  controls  the 
penetration  of  Azotobacter  organisms  as  above  portrayed.  I  think 
in  addition,  however,  it  may  fairly  be  argued  that  the  presence 
of  Azotobacter  in  more  than  half  of  these  soils  in  the  third  foot 
is,  in  itself,  a  favorable  indication  of  the  nature  of  the  soils  in 
question.  It  is  of  interest  also  that,  in  soil  No.  5,  Azotobacter 
organisms,  with  the  nitrogen-fixing  power  as  vigorous  as  those 
above,  were  found  in  the  fourth  foot.    This,  so  far  as  the  writer  is 


1912]  Lipman:  Bacteria  in  Soils  of  Arid  Eegions  15 

aware,  constitutes  the  only  published  case  of  even  this  extent  of 
penetration  of  Azofobacter  organisms.  It  has  been  reported  to 
me,  however,  that  Azobacter  organisms  have  been  found  in  the 
twelfth  foot  of  soil  in  some  of  the  very  favorably  constituted  loess 
soils  of  Nebraska.  The  question  put  in  the  introduction  to  this 
subject  of  nitrogen  fixation  is  therefore  answered  in  the  negative. 
For  the  greater  depths,  at  any  rate,  in  which  ammonification 
manifestly  is  vigorous  in  our  soils,  Azotobacter  organisms  do 
not  penetrate  and  are  not  the  source  of  the  supply  of  nitrogen 
which  can  be  transformed  at  those  depths  by  the  ammonifying 
organisms  or  by  the  nitrifying  organisms.  The  nitrogen  supply 
of  these,  therefore,  in  the  lower  layers  of  the  soil  must  be  the 
humus  produced  from  the  decaying  roots  at  those  depths,  or 
the  humus  brought  down  in  solution  from  the  upper  layers  of 
the  soil. 

As  regards  soil  No.  6  we  have  here  again  a  total  absence 
of  Azotobacter  organisms,  possibly  due  in  part,  at  least,  if  not 
wholly,  to  the  absence  of  any  but  very  small  amounts  of  humus, 
by  which  I  have  already  tried  to  explain  the  feeble  nitrification 
only  in  the  first  foot  of  this  same  soil.  The  same  remarks 
also  which  were  made  above,  with  reference  to  soil  Nos.  8,  9,  and 
10,  as  regards  their  ammonifying  and  particularly  their  nitrify- 
ing power,  apply  again  in  the  case  of  their  nitrogen-fixing  power. 
No  Azotobacter  organisms  and  no  fixation  of  nitrogen  were  ever 
observed  in  any  of  these  soils,  no  matter  from  what  depth  of  soil 
the  cultures  were  prepared. 

In  justice  to  this  subject  it  must  further  be  stated  here  that 
the  comparatively  slight  penetration  of  Azotobacter  organisms 
in  our  soils  may  be  due  to  factors  other  than  merely  a  lack  of 
a  plentiful  supply  of  oxygen.  There  is  evidently  some  other 
circumstance  which  controls  the  presence  or  absence  in  many 
of  our  soils  of  Azotobacter  organisms  and  that  may  also  limit 
the  depth  to  which  these  organisms  may  penetrate.  Just  what 
this  factor  may  be  is  not  at  present  clear  to  the  writer,  but  the 
fact  remains  that  frequently  soils  with  a  good  chemical  and 
physical  constitution  and  producing  good  crops,  will  yet  show 
no  Azotobacter  organisms. 


16  University  of  California  Puhlications  in  Agricultural  Sciences    [Vol.  1 

GENERAL  DISCUSSION 

As  I  have  already  pointed  out  in  an  earlier  publication,^  the 
slow  formation  of  clay  substances  in  soils  of  the  arid  region, 
owing  to  the  peculiar  climatic  conditions  there  obtaining,  is 
doubtless  responsible  for  a  much  greater  degree  of  aeration  in 
soils  because  of  the  larger  volume  of  pore  spaces  made  possible 
through  a  lack  of  large  quantities  of  cementing  substances.  Thus 
when  soils  first  begin  to  form  from  disintegrating  rock  we  have 
much  more  complete  aeration  with  an  encouragement  for  bac- 
teria, probably  the  earliest  inhabitants  of  the  soil,  to  penetrate 
to  greater  depths.  Such  penetration  on  the  part  of  bacteria  is 
invariably  accompanied  by  the  production  of  more  favorable 
physical  and  chemical  conditions  in  the  soil  for  the  roots  of 
plants.  These  in  their  turn,  through  physical  and  chemical 
changes  which  they  bring  about  in  the  soil  in  their  search  for 
water  and  food,  make  better  conditions  for  a  deeper  penetration 
of  bacteria  and  so  through  mutual  aid  the  latter  and  the  higher 
plants  are  able,  under  our  arid  climatic  conditions,  to  make  the 
deeper  layers  of  soil  a  more  congenial  medium  for  each  other. 
The  changes  thus  brought  about  result  in  a  more  uniform  tex- 
ture of  soils  at  great  depths,  uniformity  of  chemical  composi- 
tion, including  humus  content,  in  all  the  soil  layers,  and  a 
much  closer  approximation  of  the  bacterial  flora  in  the  lower  soil 
layers  to  those  of  the  upper  layers  than  can  be  found  in  the 
average  soils  of  the  humid  region,  where  climatic  conditions  are 
unfavorable  to  good  aeration,  because  tendencies  opposite  to  those 
above  described  for  our  soils  are  in  operation.  An  estimate  of 
the  biological  condition  of  our  deep  soils  was  thus  similarly  made 
by  Hilgard  on  a  priori  considerations  and  the  investigations 
above  recorded  serve,  in  general,  to  confirm  his  surmise. 

Viewing  the  subject  in  its  entirety,  we  find  that  the  organ- 
isms forming  ammonia  in  soils  penetrate  to  greater  depths  than 
the  nitrifying  or  nitrogen-fixing  bacteria  studied.  While  am- 
monification  is  usually  most  vigorous  in  the  surface,  four  to  six 
feet,  it  is  none  the  less  very  pronounced  in  the  lower  layers  from 
six  to  ten  feet  in  depth  in  all  of  our  normal  deep  soils.    Hardpan, 


-  Ijii)rnan,  C.  B.,  New  Facts  about  Bacteria  of  California  Soils,  Science 
N.  S.,  June  11,  1909. 


1912]  Lipman :  Bacteria  in  Soils  of  Arid  Regions  17 

alkali,  and  a  lack  of  humus  and  moisture  decrease  the  ammoni- 
fying powers  of  our  soils  or  are  not  favorable  to  the  develop- 
ment of  vigorous  ammonifying  flora,  but  their  effects  are  just  as 
pronounced  in  the  upper  layers  of  these  abnormal  soils  as  in 
the  lower  layers  which,  therefore,  cannot  be  fairly  compared 
with  our  deep  average  soils  as  to  bacterial  content.  To  what  a 
serious  extent  alkali  salts  may  affect  ammonification  has  been 
shown  by  me  in  a  recent  paper.^  That  the  humus  content  alone 
may  profoundly  affect  the  number  and  vigor  of  bacteria  is  well 
exemplified  in  both  soils  No.  6  and  10,  where  all  other  condi- 
tions but  the  humus  content  are  favorable  and  where  both  the 
number  and  physiological  efficiency  of  the  organisms  is  small. 

It  would  therefore  seem,  in  brief,  that  ammonification  is 
vigorously  active  in  the  lower  soil  layers  in  soils  of  the  arid  region 
where  humus  is  present  and  hardpan  and  alkali  are  absent. 
Since  these  conditions  are  complied  with  in  the  average  of  our 
cropped  soils,  the  opinion  is  justified  that  the  deep  penetration 
of  bacteria  is  a  distinctive  characteristic  of  soils  in  arid  regions 
which  results  from  much  better  aeration,  as  a  starting  point, 
than  can  be  attained  in  soils  of  the  humid  region.  The  experi- 
mental data  above  given  amply  confirm  this  opinion  and  help 
to  explain  why  deep  plowing  is  not  only  harmless  in  our  soils 
but  directly  beneficial,  and  why  three  or  four  feet  of  upper  soil 
may  be  removed  in  grading,  and  alfalfa  and  fruit  trees  may  be 
grown  on  the  newly  uncovered  subsoil  without  difficulty,  a  feat 
which  cannot  be  accomplished  on  soils  of  the  humid  regions. 

As  for  nitrification  my  data  present  again  features  of  striking 
interest.  They  go  to  prove  that  nitrate  formation,  like  ammoni- 
fication, goes  on  at  much  greater  depths  in  soils  of  arid  than  in 
soils  of  the  humid  region,  and  thus  render  distinctly  sectional  the 
observations  of  Dyer*  on  this  subject,  and  makes  them  applicable 
only  to  soils  of  the  humid  region.  While  the  nitrifying  organ- 
isms are  doubtless  more  susceptible  to  a  lack  of  oxygen  than  the 
ammonifying  bacteria,  the  differences  obtained  above  between 
the  two  groups  of  organisms,  so  far  as  soil  fertility  is  concerned, 
are  rather  those  of  degree  than  of  kind.     The  same  relationships 


4  Bulletin  106,  p.  55,  O.  E.  S.,  U.  S.  D.  A. 
3  Centrallblatt  fiir  Bakt.,  2  Abt.,  vol.  32,  p.  58. 


18  University  of  California  Publications  in  Agricultural  Sciences    [Vol.  1 

displayed  by  the  ammonifying  bacteria  toward  hardpan,  alkali, 
and  a  lack  of  humus  and  moisture,  hold  in  a  more  exaggerated 
way  as  regards  the  nitrifying  organisms.  More  specifically,  the 
writer  has  also  shown^  the  distinct  effects  of  each  of  the  alkali 
salts  on  nitrifying  bacteria  in  work  quite  recently  completed.  It 
would  appear  in  general,  however,  that  in  our  deep  soils,  a  supply 
of  nitrate  as  well  as  of  ammonia  is  at  the  disposal  of  plants  for 
a  depth  of  five  or  six  feet.  As  regards  the  nitrogen-fixing  powers 
of  soils  of  the  arid  region,  my  results  show  plainly  that  they  do 
not  differ  strikingly  from  those  of  soils  in  the  humid  regions, 
if  the  presence  and  vigor  of  Azotobacter  organisms  be  taken  as 
a  criterion.  While  it  is  true  that  in  one  or  two  cases  Azotobacter 
organisms  were  found  in  our  soil-columns  below  the  depth  at 
which  they  occur  elsewhere,  and  perhaps  at  a  slightly  greater 
depth  in  all  soils  in  which  they  were  found,  I  feel  loath  to  believe 
that  these  are  expressions  of  a  rule  for  soils  of  the  arid  region. 
Other  observations  indeed  lead  me  to  believe  that  Azotobacter 
development  has  not  gone  so  far  in  our  soils  as  it  has  in  soils 
of  other  regions.  For  example,  I  have  studied  many  soils  in  Cali- 
fornia with  a  favorable  physical  and  chemical  constitution  which 
were  absolutely  devoid  of  Azotobacter  organisms.  If  therefore 
the  results  set  forth  above,  with  reference  to  nitrogen  fixation, 
are  to  be  considered  representative,  the  nitrogen  supply  in  the 
lower  layers  of  the  soil  must  be  replenished  in  this  region  as 
well  as. in  the  humid  region,  not  from  direct  fixation  by  Azoto- 
bacter, but  from  the  nitrogen  of  the  upper  soil  layers. 

With  reference  to  these  investigations  in  general,  one  or  two 
additional  points  need  more  than  passing  consideration.  First, 
as  to  the  method  of  collecting  the  soil-samples  for  examination, 
it  appears  to  the  writer  that  every  possible  precaution  was  used 
to  prevent  contamination  and  it  would  be  difficult  to  devise  a 
method  which  takes  into  consideration  and  avoids  more  of  the 
avenues  of  contamination  by  which  any  results  might  be  vitiated. 
Moreover,  I  find  strong  confirmation  of  this  belief  in  the  facts 
brought  out  in  the  data  above  given,  viz.,  that  any  abnormality 
in  the  soil  was  sure  to  be  reflected  in  the  results  obtained  with 
cultures  prepared  from  that  abnormal  soil.    Thus  hardpan  layers 


5  Cent,  f  iir  Bakt.,  2  Abt.,  vol.  33,  p.  305. 


1912]  TApman  :  Bacteria  in  Soils  of  Arid  lief/ionn  19 

never  ^^ave  evidence  of  vigorous  bacteria,  nor  did  alkali  soils 
or  soils  devoid  of  humus. 

Secondly,  the  writer  desires  to  anticipate  criticism  on  the 
method  used  in  culturin^i'  the  organisms  of  the  various  soil 
samples,  viz.,  a  modified  Remy  sohition  method.  No  one  is  more 
ready  than  I  am  to  admit  the  just  criticism  made  of  the  solu- 
tion-culture methods  in  soil  bacteriology.  Indeed  I  believe  that 
I  was  one  of  the  first  to  put  into  practice  on  a  lar^ic  scale  the 
direct  soil-culture  method  in  the  laboratory.  But  when  problems 
of  the  nature  involved  in  these  investigations  must  be  attacked, 
regard  must  be  had  for  the  chances  of  contamination  in  the 
method  employed,  and  for  the  feasibility  of  obtaining,  uncon- 
taminated,  large  volumes  of  soil  for  use  in  these  experiments. 
When  these  were  considered  from  all  points  of  view,  only  one 
feasible  and  reliable  method  of  culturing  the  soils  seemed  avail- 
able and  that  was  the  solution  method.  The  difficulties,  prac- 
tically insurmountable,  which  must  arise  with  any  other  method, 
when  such  work  is  carried  out  on  a  large  scale  as  it  must  of 
necessity  be,  can  be  fully  appreciated  by  those  who  have  ever 
attempted  it.  The  gratifying  results  obtained  in  this  work,  how- 
ever, seem  to  me  a  further  justification  of  the  methods  em- 
ployed. 

It  seems  of  particular  moment  now,  to  call  the  attention  of 
soil  bacteriologists  in  particular,  and  soil  scientists  in  general, 
to  the  important  field  explored  in  these  investigations  and  the 
striking  results  obtained  therefrom,  not  only  because  it  repre- 
sents a  new  field  of  research,  but  because  it  emphasizes  more 
strongly  than  ever  the  radical  differences  which  obtain  between 
soils  of  the  humid  and  arid  regions.  It  also  helps  to  explain 
the  extraordinary  appearance  of  our  subsoils  (if  subsoils  they  be) 
and  the  marvellous  root  developments  of  which  plants  under  our 
climatic  conditions  are  capable.  While  these  studies  have  not  yet 
departed  from  the  realms  of  the  preliminary,  they  are  replete 
with  facts  which  are  already  of  considerable  practical  and  scien- 
tific significance  and  which  are  doubtless  destined  to  become 
more  so  as  time  progresses.  As  a  part  especially  of  a  comprehen- 
sive soil  study  they  are  invested  with  unusual  importance  and 
may  help  to  solve  problems  now  perplexing  and  difficult  to  study. 


20  University  of  California  Publications  in  Agricultural  Sciences    [Vol.  1 


CONCLUSIONS 

Investigations  of  the  distribution  and  activities  of  bacteria  in 
soils  of  the  arid  region  show: 

1.  That  samples  of  soil  for  studying  the  flora  of  each  layer 
of  soil  can  best  be  obtained  from  a  hole  twelve  feet  in  depth 
with  at  least  one  vertical  wall,  the  latter  when  sterilized  being 
sampled. 

2.  That  tin  tubes  ten  inches  long  and  about  one  inch  in 
diameter  closed  at  one  end  and  cotton-stoppered  are  best  for 
collecting  the  samples. 

3.  That  the  solution  method  for  stud3ang  the  soils,  despite 
its  many  drawbacks,  is  the  most  feasible  one  to  emplo3^ 

4.  That  soils  of  the  arid  region  at  all  depths  studied  show 
ammonifying  powers  which,  however,  are  generally  most 
vigorous  in  the  first  six  or  eight  feet.  In  one  case  ammonifica- 
tion  was  noted  in  soil  from  a  depth  of  fifteen  feet,  or  adjoining 
the  water-table. 

5.  That  nitrification  is  found  commonly  down  to  a  depth  of 
five  to  six  feet  in  soils  of  the  arid  region.  In  one  case  soil  from 
the  eight-foot  depth  showed  a  vigorous  nitrifying  power. 

6.  That  nitrogen  fixation  through  Azotobatcer  does  not  go 
on  below  two  feet  in  the  soil  usually,  but  has  been  found  in 
some  soils  at  three  feet  and  in  one  soil  down  to  four  feet.  Many 
soils  in  the  arid  region,  otherwise  favorably  constituted,  do  not 
contain  Azotobacter  organisms. 

7.  That  from  the  point  of  view  of  ammonification  and  nitri- 
fication soils  in  the  arid  region  differ  markedly  from  those  in 
the  humid  region  when  the  lower  layers  of  soil  are  considered. 
The  difference  is  not  marked  as  regards  nitrogen  fixation. 

8.  The  results  above  recorded  help  to  explain  the  favorable 
physical  and  chemical  constitution  of  our  soil  and  also  the  deep 
rooting  of  plants  so  characteristic  of  the  arid  regions. 

Transmitted  April  S,  WIS. 


